Allergen Summary: Detailed accomplishments of each project are summarized below. Sensitization to cockroach allergens is a major risk factor for asthma, especially among inner city residents. The structure of the cockroach allergen Bla g 1 was determined by X-ray crystallography. One of our stated goals has been to map where patient antibodies interact with the allergens in order to suggest modifications for immunotherapy that would generate fewer adverse symptoms in patients. We have been studying a model antibody interacting with Bla g 1, and we have been attempting to crystallize Bla g 1 in complex with this model, which would give direct evidence of the interacting epitope. Although we have been unable to crystallize the model antibody in complex with the allergen, we did crystallize the antibody alone. From this we deduced that the binding site on the antibody was highly basic, and hence we generated numerous site-directed mutants of Bla g 1 at primarily acidic sites and tested the effect on binding to the model antibody. From this data we are able to suggest a model for the antibody-interacting epitope of the Bla g 1. Cyclophilin allergens are considered pan-allergens due to their high cross-reactivity; i.e. patients sensitized to just one source are usually highly allergic to the allergens from all other sources. This cross-reactivity can include auto-reactivity where the immune system mistakenly reacts against self-antigens. Indeed, some patients with chronic allergic disease, either asthma or atopic dermatitis, have demonstrated both humoral and cell-mediated autoreactivity. The basis for the cross reactivity is believed to be the high sequence identity between members of this protein family. In this period we completed our determination of the structure of the allergen Cat r 1, derived from the rosy periwinkle using NMR techniques. This is the first structure of a cyclophilin protein derived from plants. Using the structure, we have been able to better understand the important residues that likely account for the cross reactivity between plant and mold allergens, and potential residues involved in autoreactivity with human cyclophilins. This knowledge will help in the rational design of immunotherapeutics in that researchers may now design hypo-allergens that also avoid encouraging autoreactivity. Like the cyclophilin allergens, there are GST allergens from many different species but these have received comparatively less attention. A recent paper sparked renewed interest in these allergens due to the demonstrated cross-reactivity of the GST allergen Bla g 5 with a helminth GST from Wucheria bancrofti. A connection between the immune response to helminthes and allergens has long been suspected because humans respond to both with same antibody subtype, IgE. We investigated the cross-reactivity further by determining the structures of Bla g 5, Der p 8, and Blo t 8, all GST-allergens. Further, we compared the cross reactivity of these allergens with a GST from another helminth, Ascaris sp., which we suspected may have similar cross reactivity to the other GST allergens. Using the structures we compared surface exposed residues and correlate the information with patient cross-reactivity. Despite published reports of cross-reactivity among GST allergens from patients in tropical countries there was very little cross-reactivitiy found in patients from N. Amercia, a more temperate biome, where the predominant GST allergen sensitizers are Bla g 5 and Der p 8. This result is substantiated by comparing the surface residues of the structures of all these allergens. There are very few regions where there is significant residue identity. This information is useful for clinicians. It informs them that N. American patients sensitized to Bla g 5 or Der p 8 are unlikely to be sensitive to the allergens from tropical species of mites or helminths. The protein Ara h 2 is the most potent peanut allergen recognized by >90% of peanut allergic patients. The natural allergen and the recombinant construct used to determine the structure showed different patterns of recognition by patient sera. Based on these comparisons a major site of interaction (an epitope) for about 50% of patients was identified. This success has encouraged us to further map the patient epitopes using a panel of antibodies with various specificities for Ara h 2 and the homologous Ara h 6 allergen. Currently we have selected and produced the Fab fragment of several antibodies and are currently performing crystallization trials with complexes of Ara h 2. Thus far, we have failed to generate quality crystals. We plan to attempt new strategies for crystallization including C-terminal affinity/crystallization tags for Ara h 2. Another problem is the expense of production of the antibodies in hybrodomas for the quantities needed for crystallization. Over the past year the genes for the antibodies were clones and we have created recombinant expression systems in E. coli that are being tested for production. It is our goal to further identify conformational epitopes on peanut allergens in order to better understand the patient response to peanut and to determine whether specific epitope recognition correlates with any aspect of peanut allergic disease, e.g. risk of anaphylaxis, emergency room visits, or response to oral therapy. Similar to the study above, we have generated an ScFv expression system for the anti-Der p 7 antibody WH9. This antibody blocks up to 60% of the patient response to the important dust mite allergen. Improved understanding the epitopes of the complex with Der p 7 will be facilitate better understanding the human response, with the hope of generating future therapeutics. Several previous studies, including those from our group, have identified the human protein RAGE as potentially important in the pathway of sensitization to allergens. We previously demonstrated that RAGE specifically binds to peanut allergens Ara h 1 and Ara h 3 after the peanuts have been roasted. It has been proposed that the process of dry roasting contributes to sensitization and this provided direct mechanistic evidence that this was possible via the RAGE receptor. Further research is proceeding in two directions. First, the modifications to the peanut allergens are being more carefully categorized by mass spectroscopy. This approach required a number of technical innovations, but the system is now working well and we are beginning to categorize the modifications in large numbers. Second, it is fascinating that RAGE is capable of recognizing the diverse array of chemical modifications from cooking that we are discovering. We are attempting to synthesize specific modifications on peanut allergen-derived peptides and further trying to characterize how these modifications interact with RAGE. Hopefully, this approach can shed light on the basis for the apparent ligand promiscuity of this receptor, which may lead to new approaches for inhibiting or enhancing this response in patients, depending on the desired outcome.